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2 b-lactamaseClinically isolated distinct b-lactamase now number > The simplest classification is by protein sequence, four classes, A, B, C, and D, Classes A, C, and D include enzymes that hydrolyze their substrates by forming an acyl enzyme through an active site serine, whereas class B b-lactamases are metalloenzymes that utilize at least one active-site zinc ion to facilitate b-lactam hydrolysis.b-lactamaseClinically isolated distinct b-lactamase now number > 1300.The simplest classification is by protein sequence, whereby the -lactamases are classified into four molecular classes, A, B, C, and D, based on conserved and distinguishing amino acid motifs (2, 3, 29, 46). Classes A, C, and D include enzymes that hydrolyze their substrates by forming an acyl enzyme through an active site serine, whereas class B -lactamases are metalloenzymes that utilize at least one active-site zinc ion to facilitate -lactam hydrolysis. Although a structural approach is the easiest and least controversial way to classify such a diverse set of enzymes, a functional classification provides the opportunity to relate these varied enzymes to their clinical role, i.e., by providing selective resistance to different classes of -lactam antibiotics. Functional groupings, admittedly, can be more subjective than structural classes, but they aid the clinician and laboratory microbiologist in correlating the properties of a specific enzyme with the observed microbiological resistance profile for a clinical isolate.

5 Carbapenemases are β-lactamases that hydrolyze penicillins, in most cases cephalosporins, and to various degrees carbapenems and monobactams (the latter are not hydrolyzed by metallo-β-lactamases).

6 b-lactamase ClassA enzymesClassA enzymes are very prevalent among bacteria, especially in the Enterobacteriaceae. TEM-1 and TEM-2 were some of the first beta- lactamases described in the 1960s. SHV-1 was discovered later. These enzymes can be encoded on conjugative plasmids, which have served as genetic vehicles for wide dispersal of these resistance genes. SHV-1 is also found on the chromosome of Klebsiella pneumoniae. TEM-1, TEM-2, and SHV-1 confer resistance to commonly used antibiotics, such as ampicillin and amoxicillin. Other important substrates for these three enzymes include carbenicillin, pipercillin, cefazolin, and cefuroxime. The extent of hydrolysis for these drugs can vary depending upon the quantity of the b- lactamase that is produced. Class A enzymes are inhibited by clavulanic acid (sulbactam, tazobactam). In addition, clavulanic acid has proved useful in the laboratory for detection of some classA enzymes.ClassA enzymes are very prevalent among bacteria, especially in the Enterobacteriaceae. TEM-1 and TEM-2 were some of the first beta-lactamases described in the 1960s. SHV-1 was discovered later. These enzymes can be encoded on conjugative plasmids, which have served as genetic vehicles for wide dispersal of these resistance genes. SHV-1 is also found on the chromosome of Klebsiella pneumoniae. TEM-1, TEM-2, and SHV-1 confer resistance to commonly used antibiotics, such as ampicillin and amoxicillin. Other important substrates for these three enzymes include carbenicillin, pipercillin, cefazolin, and cefuroxime. The extent of hydrolysis for these drugs can vary depending upon the quantity of the beta-lactamase that is produced (2). ClassAenzymes are inhibited by clavulanic acid. In addition, clavulanic acid has proved useful in the laboratory for detection of some classA enzymes.

7 b-lactamase ClassA ESBL enzymesESBLs hydrolyze penicillins, narrow- and extended-spectrum cephalosporins (including the anti-methicillin-resistant S. aureus [MRSA] cephalosporin), and the monobactam aztreonam. In contrast, ESBLs cannot efficiently degrade cephamycins, carbapenems, and b–lactamase inhibitors. More than 200 different ESBLs have been identified, posing a significant risk to public health and to hospitalized patients in intensive care units, where infection with an ESBL may lead to significant morbidity and mortality The majority of ESBLs are from the SHV, TEM, and CTX-M families; less frequently they are derived from BES, GES-1, VEB, and PER enzymes, and sometimes these enzymes do not belong to any defined family.ClassA enzymes are very prevalent among bacteria, especially in the Enterobacteriaceae. TEM-1 and TEM-2 were some of the first beta-lactamases described in the 1960s. SHV-1 was discovered later. These enzymes can be encoded on conjugative plasmids, which have served as genetic vehicles for wide dispersal of these resistance genes. SHV-1 is also found on the chromosome of Klebsiella pneumoniae. TEM-1, TEM-2, and SHV-1 confer resistance to commonly used antibiotics, such as ampicillin and amoxicillin. Other important substrates for these three enzymes include carbenicillin, pipercillin, cefazolin, and cefuroxime. The extent of hydrolysis for these drugs can vary depending upon the quantity of the beta-lactamase that is produced (2). ClassAenzymes are inhibited by clavulanic acid. In addition, clavulanic acid has proved useful in the laboratory for detection of some classA enzymes.

8 b-lactamase Class A serine carbapenemasesClass A serine carbapenemases include the nonmetallocarbapenamase of class A (NMC-A), IMI, SME, and KPC. Members of this group of b- lactamases can hydrolyze carbapenems as well as cephalosporins, penicillins, and aztreonam. These carbapenemhydrolyzing enzymes have been identified primarily in Enterobacter cloacae, Serratia marcescens, and K. pneumoniae, bacteria which often harbor multiple resistance determinants, narrowing the range of treatment options. The bla gene for the former two organisms is typically found on the chromosome, while the K. pneumoniae carbapenemase blaKPC gene is carried on plasmids containing Tn MICs of carbapenems in carbapenemase-expressing strains can vary from moderately increased (2 to 4 g/ml) to resistant ( 32g/ml)ClassA enzymes are very prevalent among bacteria, especially in the Enterobacteriaceae. TEM-1 and TEM-2 were some of the first beta-lactamases described in the 1960s. SHV-1 was discovered later. These enzymes can be encoded on conjugative plasmids, which have served as genetic vehicles for wide dispersal of these resistance genes. SHV-1 is also found on the chromosome of Klebsiella pneumoniae. TEM-1, TEM-2, and SHV-1 confer resistance to commonly used antibiotics, such as ampicillin and amoxicillin. Other important substrates for these three enzymes include carbenicillin, pipercillin, cefazolin, and cefuroxime. The extent of hydrolysis for these drugs can vary depending upon the quantity of the beta-lactamase that is produced (2). ClassAenzymes are inhibited by clavulanic acid. In addition, clavulanic acid has proved useful in the laboratory for detection of some classA enzymes.

9 b-lactamase Class B metallo-b-lactamasesClass B enzymes are Zn- dependent b-lactamases that demonstrate a hydrolytic mechanism different from that of the serine b-lactamases of classes A, C, and D. Organisms producing these enzymes usually exhibit resistance to penicillins, cephalosporins, carbapenems, and the clinically available b-lactamase inhibitors . Interestingly, the hydrolytic profile of MBLs does not typically include aztreonam. MBLs likely evolved separately from the other Ambler classes, which have serine at their active site. The blaMBL genes are located on the chromosome, plasmid, and integrons . P. aeruginosa, K. pneumoniae, and A. baumannii produce class B enzymes encoded by mobile genetic elements. In contrast, Bacillus spp., Chryseobacterium spp., and Stenotrophomonas maltophilia possess chromosomally encoded MBLs, but the majority of these pathogens are generally not responsible for serious infections.ClassA enzymes are very prevalent among bacteria, especially in the Enterobacteriaceae. TEM-1 and TEM-2 were some of the first beta-lactamases described in the 1960s. SHV-1 was discovered later. These enzymes can be encoded on conjugative plasmids, which have served as genetic vehicles for wide dispersal of these resistance genes. SHV-1 is also found on the chromosome of Klebsiella pneumoniae. TEM-1, TEM-2, and SHV-1 confer resistance to commonly used antibiotics, such as ampicillin and amoxicillin. Other important substrates for these three enzymes include carbenicillin, pipercillin, cefazolin, and cefuroxime. The extent of hydrolysis for these drugs can vary depending upon the quantity of the beta-lactamase that is produced (2). ClassAenzymes are inhibited by clavulanic acid. In addition, clavulanic acid has proved useful in the laboratory for detection of some classA enzymes.

10 b-lactamase Class C serine cephalosporinasesClass C AmpC b–lactamases include CMY-2, P99, ACT-1, and DHA-1, which are usually encoded by chromosomal bla genes, although plasmid- borne AmpC enzymes are becoming more prevalent. Organisms expressing the AmpC b–lactamase are resistant to penicillins, b-lactam–b- lactamase inhibitor combinations, and cephalosporins, including cefoxitin, cefotetan, ceftriaxone, and cefotaxime. AmpC enzymes poorly hydrolyze cefepime and are inhibited by cloxacillin, oxacillin, and aztreonam. Members of the Enterobacteriaceae family, such as Enterobacter spp. and Citrobacter spp., are AmpC b-lactamase producers that resist inhibition by clavulanate and sulbactam, although Klebsiella spp., Salmonella spp., and Proteus spp. normally do not harbor chromosomal blaAmpC genes. Production of chromosomal AmpCs in Gram-negative bacteria is at a low level (“repressed”) but can be “derepressed” by induction with certain b- lactams, particularly cefoxitin.

11 b-lactamase Class D serine oxacillinasesClass D b-lactamases were initially categorized as “oxacillinases” because of their ability to hydrolyze oxacillin at a rate of at least 50% of that of benzylpenicillin, in contrast to the relatively slow hydrolysis of oxacillin by classes A and C. In bacteria, OXA b–lactamases can also confer resistance to penicillins, cephalosporins, extended-spectrum cephalosporins (OXA- type ESBLs), and carbapenems (OXA-type carbapenemases). OXA enzymes are resistant to inhibition by clavulanate, sulbactam, and tazobactam, (with some exceptions; e.g., OXA-2 and OXA-32 are inhibited by tazobactam but not sulbactam and clavulanate, and OXA-53 is inhibited by clavulanate). Examples of OXA enzymes include those rapidly emerging in A. baumannii (e.g., OXA-23 and OXA-24/40) and constitutively expressed in P. aeruginosa (e.g., OXA-50).-

17 Avibactam DBO diazabiciclo octanoniAvibactam has an extremely broad spectrum of activity against classes A and C serine b-lactamases, including ESBLs and class A carbapenemases This molecule inhibits selected class D b-lactamases including OXA-48, but apparently not other class D carbapenemases, as judged by the absence of synergy with imipenem against resistant strains of A. baumannii producing OXA-51 and OXA-58 and does not inhibit class B MBLs.

20 Aztreonam-AvibactamMonobactams are hydrolyzed by ESBLs but are inherently stable to hydrolysis by MBLs, an avibactam- aztreonam combination resulted inhibitory to MBL- producing pathogens.Monobactams are hydrolyzed by ESBLs but are inherently stable to hydrolysis by MBLs, an avibactam-aztreonam combination resulted inhibitory to MBL-producing pathogens. In a study of carbapenemase-producing Enterobacteriaceae, aztreonam had MICs of 4 g/ml in combination with 4 g/ml avibactam, including strains that produced OXA-48, serine carbapenemases, or MBLs When the aztreonam-avibactam combination was tested against 126 P. aeruginosa isolates, aztreonam tested as resistant for 27.8% of the isolates, compared to 1.6% of the isolates that were resistant to a ceftazidime-avibactam combination This suggests that the ceftazidime combination would be preferable against non-MBL-producing pseudomonal isolates.

21 MK 7655MK-7655, a new member of the DBO series. A combination of imipenem and MK-7655 has excellent activity against a KPC- 2-producing isolate of K. pneumoniae and displays moderate improvements in the imipenem efficacy against most AmpC-overexpressing isolates of P. aeruginosa.

23 RPX7009RPX7009, a boronic acid-containing - lactamase inactivator with inhibitory activity against class A and class C serine -lactamases, particularly highlighting both in vitro and in vivo activity against KPC-producing K. pneumoniae. Boronic acid inhibitors of PBPs and of classes A, C, and D - lactamases had previously been identified, but none has achieved success as a clinical candidate. Preclinical testing of RPX7009 indicated that the inhibitor had no off-target effects, and it was well tolerated at high doses, with no safety signals that would preclude future development

24 Biapenem-RPX7009Biapenem (“RPX- 2003”) is a broad-spectrum carbapenem with in vitro activity against Gram-negative and Gram-positive bacteria similar to that of meropenem. Like other carbapenems, biapenem is not affected by the presence of ESBLs but is labile to hydrolysis by both serine and metallo-carbapenemases. Although carbapenem resistance is increasing, 75% of recent Japanese pseudomonal isolates were susceptible to biapenem and meropenem. Pharmacologically, biapenem is notable for its low proconvulsive activity compared to that of imipenem

25 Carbapenem + carbapenemA combination of ertapenem and doripenem in both an in vitro chemostat and an in vivo murine thigh infection model. Overall, the combination of doripenem plus ertapenem demonstrated enhanced efficacy over either agent alone (2011) Double-Carbapenem Therapy Not Proven To Be More Active than Carbapenem Monotherapy against KPC-Positive Klebsiella pneumoniae (2012) Ertapenem plus doripenem or meropenem were given in three patients suffering from pandrug-resistant, KPC-2-positive Klebsiella pneumoniae bacteremia (2 patients) and urinary tract infection (1 patient), respectively. All responded successfully, without relapse at follow-up. The results obtained should probably be attributed to ertapenem’s increased affinity for the carbapenemases hindering doripenem/meropenem degradation in the environment of the microorganism (2013)